Nuclear transport is critical for the maintenance of the levels and activities of transcription factors, nuclear kinases, and replication factors. We identified Ca+2/calmodulin as an activator of nuclear import and suggested a role for Ca+2 in the regulation of nuclear import during cell activation. We also demonstrated a role for the multifunctional lectin calreticulin in nuclear export. We suggest that bi-directional transport across the nuclear pore is controlled by GTP and Ca+2, thus providing coordinate regulation of nuclear transport and other signal transduction pathways. We are currently examining the mechanism by which Calreticulin may be differentially targeted to the endoplasmic reticulum and the nucleus and its role in modulating nuclear export. Posttranslational modifications are another means by which gene regulation may be regulated. O-GlcNAc is an intracellular glycan modification of Ser/Thr proposed to participate in diverse signaling pathways, via competition with phosphorylation. We seek to understand the biological functions of O-GlcNAc-dependent signaling and to determine whether altered O-GlcNAc metabolism contributes to human diseases such as diabetes mellitus and neurodegeneration. Addition and removal of O-GlcNAc are catalyzed by O-linked GlcNAc transferase (OGT) and O-GlcNAcase, respectively. The best-characterized cellular targets modified by O-GlcNAc are nuclear pore complexes (NPC) and transcription complexes. The NPC mediates macromolecular traffic across the nuclear membrane, and in metazoans, the NPC is extensively modified by O-GlcNAc. Transcription complexes are also key targets for O-GlcNAc modification. Importantly, as with histone deacetylases, OGT is recruited to Sin3a transcription-repression complexes. Based on the targets modified by O-GlcNAc, we proposed that the enzymes of O-GlcNAc metabolism modulate nuclear transport, transcription, cell growth, and apoptosis in response to nutrient availability. O-GlcNAc is transferred to proteins from UDP-GlcNAc, a sugar nucleotide whose levels are regulated by the hexosamine biosynthetic pathway (HBP) acting as a cellular ?sensor? of nutrient availability. By integrating these signals, the HBP regulates expression of a number of gene products that include leptin. In skeleta l muscle, flux through the HBP correlates with the degree of insulin resistance. The HBP is also linked to pathways regulating cell proliferation and apoptosis; fibroblasts which cannot acetylate UDP-GlcNAc exhibit defects in proliferation, adhesiveness and resistance to apoptotic stimuli. Thus, by generating UDP-GlcNAc, the HBP may be viewed as a nutrient-sensing signaling pathway. We seek to determine how O-GlcNAc participates in this signaling cascade. We are testing the hypothesis that differentially targeted isoforms of the enzymes of O-GlcNAc metabolism mediate this glycan-dependent signaling pathway. By responding to nutrient levels, this pathway modulates gene expression, cell growth and programmed cell death. Examining the structure, targeting, and regulation of the enzymes of O-GlcNAc metabolism is our principal focus. We expressed fully functional OGT and O-GlcNAcase in E. coli. Consistent with a role as a signaling molecule, we showed that OGT modifies glycogen synthase kinase-3 and casein kinase, two enzymes regulating glycogen synthesis. For O-GlcNAcase, we found that only the long isoform containing a GCN5-like acetyltransferase domain retained catalytic activity. Mutational analysis of OGT and O-GlcNAcase allowed us to define catalytic domains. We showed that OGT isoforms are targeted to both nucleus and mitochondria. The differential localization of mitochondrial and nuclear isoforms of OGT argues that they perform unique intracellular functions in apoptosis and transcriptional repression respectively. O-GlcNAcase isoforms are also differentially targeted in cells. Elevated Glycan-dependent signaling was induced upon overexpression of OGT and this induced programmed cell death. Transgenic overexpression of an isoform of OGT in muscle and fat Induced Insulin resistance and Hyperleptinemia in mice. These data demonstrate a central role for OGT in the insulin and leptin-signaling cascades. The findings suggest a more general role for glycan-dependent signaling in nutrient sensing and the pathogenesis of Type II diabetes. Using reverse genetics, knockout, and other transgenic models we are currently exploring the role of the enzymes of O-GlcNAc metabolism in signal transduction and the pathogenesis of diabetes mellitus. We have also identified a viable Caenorhabditis elegans strain lacking OGT activity and we are currently examining the effects of the deletion on nematode development and physiology.